Dynamic three-dimensional imaging of living cells is an important measurement technology in bio-application fields. For example, a fluorescence labeling technique is known which introduces a fluorescent molecule to specific DNA in cell nuclei using a fluorescence microscope and measures changes in cells undergoing mitosis. Various observations can be made by visualizing interactions of nuclei and proteins in cells stained with fluorescent molecules. A confocal laser scanning microscope is known as one that can measure a fluorescence three-dimensional image with high resolution and high contrast. However, with a confocal laser scanning microscope, it is necessary to scan the focal point for each point of the measurement object, and thus it takes time to obtain a fluorescence three-dimensional image, and accordingly there is an inherent limitation in the measurement of phenomena with rapid change of dynamic substance and simultaneous measurement of plurality of cells with different depth positions.
There is also a need for a technique that enables simultaneous observation of not only fluorescence images but also structures such as cell nuclei and cell walls. There are a phase contrast microscope and a differential interference microscope as means of structural observation. The phase difference image is effective for shape measurement of a transparent object.
If fluorescence images and phase images can be simultaneously observed, different information can be acquired simultaneously, and thus it becomes possible to provide more detailed information in the bioreaction.
Recently, an optical microscope capable of simultaneously observing a fluorescence image and a phase difference image is commercially available, but in addition to the need to replace filters, there is a restriction that one time measurement range is limited to one point or two dimensions.
On the other hand, a digital holographic microscope (Digital Holographic Microscope; DHM) is known as a tool that enables instantaneous three-dimensional measurement. In a digital holographic microscope, three-dimensional information is acquired as hologram information, and light wave information of an object with respect to a depth position is reconstructed by performing a back propagation calculation of the light wave in a computer. The digital holographic microscope is featured to have capability of three-dimensional observation of living cells without the need for special fluorescent staining (label-free), have an auto focusing function that can arbitrarily change the focal position by computer reconstruction and have capability of quantitative phase measurement. For this reason, even in a situation where cells move in three dimensions, it is possible to obtain a focused reconstructed image automatically by the reconstruction
When digital holographic microscopy is applied to cell observation, hologram acquisition of fluorescence information is required, but in order to realize holography technology using fluorescence, there is a problem that fluorescent light is a coherent light very difficult to be interfered. In recent years, methods for making holograms from incoherent light have been proposed one after another, and research for fluorescence digital holographic microscopes (Fluorescence Digital Holographic Microscope; FDHM) has been activated around the world, but most of them include many spatial light modulators and optical elements. (for example, refer to Patent literature 1)
The hologram recording device disclosed in Patent Document 1 solves the problem that light with low coherence (for example, natural light or fluorescent light) cannot be observed, and by employing a spatial light modulator (a wave-front modulation element), the object light including the first component light and the second component light with polarizing directions thereof different to each other is made to have the wave front shape of the first component light and the wave front shape of the second component light different to each other, and generating a distribution that changes spatially and periodically, and a hologram is formed by making the first component light and the second component light interfere. As a result, it is possible to obtain a reconstructed image of the subject from a hologram recorded in one imaging using low-coherence light passing through a single light path. However, this method has a problem that it is limited to the observation of a low interference three-dimensional fluorescence image.
In addition, a fluorescence digital holographic microscope (FDHM) is suitable for targeting measurement, but has a problem of being powerless for substances that do not generate fluorescence. In addition, when performing fluorescence observation, there is also a problem that the measurement object is limited because harmful fluorescent molecules are used. Therefore, from the viewpoint of application expansion of digital holographic microscope (DHM), appearance of a multimodal digital holographic microscope capable of observing various types of information such as phase images, fluorescence images and polarization is desired.
In order to realize a multimodal digital holographic microscope, the inventors of the present invention have already prototyped a digital holographic microscope capable of two-dimensional fluorescence imaging and phase imaging, and has reconstructed a prototype from the hologram image as an experiment. (Refer to Non-patent literature 1) The manufactured microscope sequentially acquires a fluorescence image and a phase image, and does not measure a fluorescence image and a phase image simultaneously.
Hereinafter, in the present specification, the fluorescence image and the fluorescence three-dimensional image, and the phase image and the phase three-dimensional image are used in the same meaning, respectively.